Association of 25-Hydroxyvitamin D Deficiency in Pediatric Epileptic Patients.

Objective Epilepsy is a chronic neurological disorder requiring long-term therapy using antiepileptic medications. Reports have incriminated long-term antiepileptic drugs use in deficiency of vitamin D and bone diseases in all age groups. We aimed to investigate the association between serum 25-hydroxyvitamin D levels and pediatric epilepsy in Indian patients. Materials & Methods We prospectively recruited 100 pediatric epilepsy patients, on monotherapy for minimum one-year duration, and 50 age and sex matched controls. This study was carried out at Yashoda Hospital, India from 2011-2014. All cases and controls underwent tests for serum 25-hydroxyvitamin D, alkaline phosphatase, serum calcium and phosphorus levels. Results Patients with 25-hydroxyvitamin D deficiency were significantly higher among cases (45%) than controls (24%). Mean alkaline phosphatase was significantly higher in cases and mean serum calcium was significantly lower (8.3±1.5) in cases. Amongst antiepileptic drugs, carbamazepine and sodium valproate were significantly associated with 25-hydroxyvitamin D deficiency. Risk of vitamin D deficiency was highest with sodium valproate usage (odds:4.0;95%CI 1.4-11.6) followed by carbamazepine use (odds: 2.7; 95%CI 1.0-6.8). After adjustment using multiple logistic regression, antiepileptic drugs showed independent association with 25-hydroxyvitamin D deficiency (odds:2.2;95%CI 0.9-4.5). Conclusion 25-hydroxyvitamin D deficiency was significantly associated with use of carbamazepine and sodium valproate in pediatric epilepsy.


Introduction
Epilepsy is a common neurological disorder that affects all age groups. Globally, around 50 million people are affected with epilepsy, which corresponds to 0.5% of the global burden of all diseases, in developing countries (1). In India, more than 10 million people suffer with seizures and prevalence of epilepsy is one percent in our population (2). The frequency rates for children vary from 2.0 to 22.2 per 1000 (3) and nearly 50% of all epileptics suffer from their childhood (4).
other systemic diseases or with neurological deficits and controlswith any history of illness or taking vitamin D supplements were excluded in the study. Standardized questions were adapted from behavioral risk factors questionnaire by the Centers for Disease Control and Prevention (CDC) (9). Patients' data were collected through face-to-face interviews from patients' parents among cases and control subjects by a senior epileptologist. Patient medical history, type of AEDs, physical and neurological examination was done by a senior neurologist. All cases and controls underwent estimation of serum alkaline phosphatase, serum calcium and serum phosphorus.

Assessment of 25-hydroxyvitamin D
For measurement of 25-hydroxyvitamin D, 5 ml blood was collected from both cases and controls. We used chemiluminescent microparticle immunoassay (CMIA) with automated instruments for assessment of 25-hydroxyvitamin D. We considered serum levels of 25 hydroxyvitamin D < 20 ng/mL as deficiency and >20.1 ng/mL as sufficient (7).

Statistical analysis
Statistical analysis was applied by SPSS ver. 15.0 (Chicago, IL, USA) software. Mean and standard deviation (SD) were estimated for various variables. Categorical variables were expressed as proportions and chi-square test was used to study the association in proportions. The student 't' test was used to test the differences in continuous variables. We performed multiple logistic regression analysis before and after adjustment for various AEDs (Carbamazepine, clobazam, clonazepam, lamotrigine, phenobarbital, sodium valproate, topiramate). All tests were two sided and P value <0.05 was considered statistically significant.

Results
We noted boys constituted 60% in both cases and controls. Mean age was 14 yrs in cases and 14.5 yrs in controls with age range of 8-18 yrs. We found significantly higher prevalence of deficiency of 25-hydroxyvitamin D, (P<0.0001) mean alkaline phosphatase (P<0.0001) and serum calcium (P=0.01) in cases compared to Epilepsy patients require longtime antiepileptic drugs (AEDs). Long-term usage of AEDs is associated with abnormal bone mineral metabolism, osteoporosis and an increased risk of fracture (5). The main mechanism seems to be centered on vitamin D deficiency caused by AEDs in epilepsy patients (6). The structure of vitamin D is a 9,10 -seco steroid and two types are dominantly present in humans (vitaminD3and vitamin D2). In circulation, vitamin D exists as serum 25-hydroxyvitamin D and this serves as a marker of vitamin D levels in the body. Vitamin D plays a vital role in maintaining a balance between phosphorus and calcium levels as well as metabolism. Vitamin D deficiency can cause reduced bone mineralization and bone fractures (7). The aim of the present study was to investigate the circulating blood levels of serum 25-hydroxyvitamin D in pediatric epilepsy patients who were on AEDs. Very limited data is available from the Indian Subcontinent.

Material and Methods
We selected 100 consecutive pediatric epilepsy cases, with age below 18 yr and 50 age and sex matched controls. This study was carried out at Department of Neurology, Yashoda Hospital, Hyderabad, India from January 2011 to December 2014. This study was approved by Institutional Ethics Committee. Informed consent was taken from the subjects.

Definition of cases and controls
Epilepsy is a disease of the brain, which results in predisposition to have epileptic seizures. Epilepsy is diagnosed when there is occurrence of at least two unprovoked seizures, occurring at least 24 hours apart (8). Controls were recruited from healthy subjects (children of staff or volunteers) from same hospital.

Inclusion and exclusion criteria of case controls
Patients who gave history of two attacks of seizures at least 24 hours apart in their life and were on monotherapy with antiepileptic drugs for minimum one year were included. Healthy controls without any history of seizures, rickets or other bone related disorders were included in the study. Patients who were on two or more AEDs, taking vitamin D supplements, suffering from

Discussion
In our study, 25-hydroxyvitamin D (≤20 ng/mL) deficiency was significantly higher among epileptics (44%) compared to control subjects (20%), which is a constant finding noted by similar studies (6,10,11). Twenty five percent of children with epilepsy had deficiency of 25-hydroxyvitamin D (10). Serum 25-hydroxyvitamin D deficiency was 75% of children with epilepsy (12). We also found significantly lower mean calcium levels in epileptic patients compared to controls (P= 0.01), our observation was advocated by others (12)(13)(14). The mechanism underlying low levels of calcium in epileptics may be multifactorial. AEDs are associated with alterations in bone metabolism and phosphate concentration and thus a change in calcium homeostasis in the body (15). However, some studies have found no significant association of calcium levels with epilepsy (16)(17)(18). We noted mean alkaline phosphatase levels were significantly higher in epileptic patients compared to controls (P<0.0001) similar to other studies (4,15,16). Elevated alkaline phosphatase levels are associated with liver or bone disease. In the present study, high alkaline controls (Table 1). Mean serum alkaline phosphatase (P=0.001), serum calcium (P=0.0006) and mean duration of taking AEDs (P<0.0001) were significantly higher among 25-hydroxyvitamin D deficient cases compared to cases with normal 25-hydroxyvitamin D levels ( Table 2). Regarding AEDs, the percentage taking carbamazepine was 27(27%), clobazam 6(6%), clonazepam 7(7%), lamotrigine 6(%), phenobarbital 24(24%), sodium valproate 21(21%) and topiramate 9(9%). Significantly higher prevalence of 25-hydroxyvitamin D deficiency was noted with carbamazepine (P=0.04) and sodium valproate (P=0.01) usage ( Figure 1). On odds ratio analysis, we established independent association of 25-hydroxyvitamin D deficiency with AEDs use. Carbamazepine (odds:2.7;95%CI:1.0-6.8) and sodium valproate (odds:4.0;95%CI:1.4-11.6) were associated more in comparison to phenobarbital (odds:2.0; 95%CI:0.8-5.1), and topiramate (odds: 0.5;95%CI:0.1-2.4) use (Table 3). After adjustment with multiple logistic regression analysis, antiepileptic drugs use was independently associated with deficiency of 25-hydroxyvitamin D in pediatric epilepsy patients ( Table 4).  (15,16,20). We noted that among epileptics, elevated alkaline phosphatase and low serum calcium were significantly associated with deficiency of 25-hydroxyvitamin D (P=0.0001), accounted by others (12)(13)(14)21). Thus in our study the main pathogenetic mechanism seems to be based on reduced active levels of vitamin D, possibly caused by induction of hepatic cytochrome P450 enzymes by AEDs, leading to its conversion to inactive metabolites in the liver microsomes. Hypocalcemia can be due to decreased absorption from the gut secondary to the state of hypovitaminosis D. This may then trigger an increase in circulating parathyroid hormone. The secondary hyperparathyroidism then leads to an increased bone turnover leading to increased serum alkaline phosphatase levels (21). other mechanism postulated include reduced intestinal calcium absorption (phenytoin) (34), impaired response to parathyroid hormone (phenobarbitone and phenytoin), (35) hypovitaminosis K (phenytoin) (36), and calcitonin deficiency (37). The exact mechanism by which sodium valproate causes similar bone mineral metabolism abnormalities is not clear but it may be mediated by a different hepatic enzyme inhibition (38). Multi-drug therapy is associated with high risk of bone mineral metabolism abnormalities than monotherapy (24). There has been a lot of debate on whether the enzyme inducing properties of AEDs are to blame. Initial studies reported an association of reduced bone mineral density and increased fracture risk with mainly enzyme inducing AEDs (EIAEDs) (39,40). However, recent studies have found no difference between EIAEDs and Non-EIAEDs in their action on 25-hydroxyvitamin D status (41). There was a similar risk of developing vitamin D deficiency with both EIAEDs and Non-EIAEDs (42). In our study too, both EIAEDs (carbamazepine (odds 2.7;95%CI:1-0-6.8)) and Non-EIAEDs (sodium valproate (odds Vitamin D inactivation by AEDs occurs mainly by induction of hepatic enzymes and by their activation of pregnane X receptor (PXR) and steroid and xenobiotic receptors (SXR) (22,30). Activation of vitamin D (D2 and D3) occurs initially in the liver where they are hydroxylated to 25(OH) D by vitamin D hydroxylase CYP27A. The antiepileptic drug binds and activates SXR. This complex binds to RXR, which then activates the 24-hydroxylase enzyme by interacting with its vitamin D responsive element (31,32). This enzyme mediates the removal of 25-hydroxyl group from both 25-hydroxyvitamin D and 1,25 dihydroxy vitamin D. This accelerated inactivation of vitamin D causes a cascade of events to adapt to the progressive insufficiency leading to secondary hyperparathyroidism. Further hypovitaminosis D results in decreased absorption of calcium from the gut. It has a detrimental effect on bone mineralization and metabolism. The above mechanism have been attributed to AEDs which are inducers of cytochrome P450 enzyme system (phenobarbitol, phenytoin and carbamazepine) (33).The  (51). Our study had some limitations, the main inadequacy was that 25-hydroxyvitamin D levels were not measured before starting AEDs and hence we cannot categorically attribute the levels to AEDs use. Second drawback was that we did not assess bone density in both cases and controls due to both financial reasons and lack of standardized reference range for Indian children. Third, as our sample size was small, we were unable to perform multiple logistic regression analysis to study the association between individual AEDs and 25-hydroxyvitamin D levels. Strengths of our study were that we recruited cases and controls from the same hospital thus reducing difference based on ethnicity, social customs and socioeconomic status.

Table3. Odds Ratio with Antiepileptic Medication
In conclusion, this study is further reemphasis the need to create more bone health awareness among epileptic patients and health care providers. We believe that all epileptic patients should be counselled regarding calcium and vitamin D intake, exposure to sunlight and physical activity, especially before initiation of AEDs. There should be precautionary assessment especially when starting on AEDs like carbamazepine and sodium valproate, which have an increased risk of impairing bone mineral metabolism. Further multicentric prospective studies are required to evaluate the role and formulate clear guidelines on prophylactic vitamin D supplementation and other measured needed for prevention and treatment of impaired bone metabolism in epilepsy.